Two copies of the sequence listing (Copy 1 and Copy 2) and a computer readable form of the sequence listing, all on CD-ROMs, each containing the file named Paxe2x80x9400359.rpt which is 39,705,377 bytes (measured in MS-WINDOWS) and was created on Jun. 12, 2001 are herein incorporated by reference.
Two copies of Table 1 on CD-ROMs, each containing 998,830 bytes (measured in MS-WINDOWS) and all having the file name paxe2x80x9400359.txt Table all created on Jun. 12, 2001, are herein incorporated by reference.
Included in the disclosure are nucleic acid molecules representing the, genome of the bacterium Myxococcus xanthus and, in particular, to nucleic acid molecules having nucleic acid sequences corresponding to DNA replication elements, genes, promoters, and other regulatory elements found in the M. xanthus genome. Also disclosed are homologous nucleic acid molecules, complementary nucleic acid molecules, polypeptides expressed by M. xanthus gene sequences, constructs comprising M. xanthus promoters, regulatory elements and/or genes, transformed cells and organisms comprising M. xanihus promoters, regulatory elements and/or genes, primers useful for replicating all or portions of M. xanthus genes or other M. xanthus nucleic acid molecules, computer readable media comprising sets of M. xanthus nucleic acid sequences, polypeptides and oligonucleotides, collections of M. xanthus nucleic acid molecules and methods of using such molecules and sequences including the use of collections of nucleic acid molecules in gene identification and gene expression analysis, development of a stoichiometric metabolic model, and preparation of constructs.
Myxococcus xanthus is a Gram-negative, rod-shaped bacterium with gliding motility that is classified within the delta subgroup of bacteria. It is a member of a group of microorganisms, commonly called myxobacteria, that generally survive by degrading organic material and other organisms in the soil. Of particular interest within the myxobacteria is the social behavior among cells. Myxobacteria form social interactions that facilitate feeding and, when nutrients become scarce, sporulation. They are the only bacteria that practice both types of social behavior, and the mechanism of communication among cells has been the subject of much research (see Myxobacteria II. 1993. Martin Dworkin and Dale Kaiser (ed.), American Society for Microbiology, Washington, D.C.). M. xanthus has been particularly well studied, and is the member of the myxobacteria with the firmest genetic and physical map on which to build a genome project. An ordered YAC library and physical map of the M. xanthus genome have been constructed (He et al., Proc Natl Acad Sci USA. 91:9584-9587 (1994); Kuspa et al., Proc Natl Acad Sci U S 91:8917-8921 (1989)). The circular genome has been estimated to be around 9.5 Mbp (Shimkets, xe2x80x9cThe Myxobacterial Genome,xe2x80x9d in Myxobacteria II. American Society for Microbiology, Dworkin and Kaiser (eds.), Washington, D.C., pp. 85-107 (1993)), which is quite large for a bacterial genome. It also has a very high G+C content (around 70%: Kaiser et al., Ann. Rev. Microbiol. 33:595-639 (1979)) which makes sequencing and assembly of the genome a significant technical challenge.
Ecology and Life Cycle of M. xanthus 
Myxobacteria are predatory organisms that can attack and degrade many other types of bacteria. Whole colonies of myxobacteria generally migrate together (swarm), and the combined production of extracellular enzymes allows more efficient solubilization of nutrients. Motility is accomplished by gliding, but the mechanism of gliding motility is not understood, either for myxobacteria or other types of gliding bacteria. The cells continue to feed communally until nutrients have been exhausted. Once nutrients become limiting, myxobacteria initiate a complex developmental process that leads to the production of fruiting bodies containing myxospores. Myxospores are resistant to heat, desiccation and other environmental insults, and serve as the resting phase for myxobacteria. The myxospores remain dormant until nutrients are again available, at which point they germinate to produce a new swarm of motile cells.
The sporulation process requires aggregation of many cells to an area where the fruiting body will eventually form. Both aggregation and fruiting body formation require a complex set of cell-to-cell communication networks, and a series of genetic switches within individual cells. The genetic cascade leads to differentiation of certain cells within the fruiting body, thereby producing myxospores.
The cells initially form a small, translucent mound. A portion of the cells within the mound begin to develop into myxospores, and the fruiting body eventually becomes about 0.1 mM high and dark as the thick spore walls are formed. The spores allow M. xanthus to survive harsh conditions for a long period of time, thus allowing the cells to be safely transported to a new location, perhaps by wind or within the gut of an animal.
Genetic analyses have identified a series of Myxococcus regulatory mutants that are defective in fruiting body formation. These mutants terminate at various points along the developmental pathway, and have defined four different chemical signaling factors, designated A, B, C, and D, that are required for normal sporulation (Kroos et al., Genes and Development 1:840-854 (1987); Losick et al., Scientific American. 276:68-73 (1997); Lee et al., J. Bacteriol. 178:977-984 (1996); Munoz et al., Microbiologia Madrid. 11:429-438 (1995); Kim, Trends in Genetics. 7:361-365 (1991)). Factors A and C are the best studied. A-factor is required for aggregation of the cells. It is actually a combination of factors, including a heat stable component that appears to be a complex mixture of amino acids (Kuspa et al., J. Bacteriol. 174:3319-3326 (1992)) and a heat labile portion that includes a mixture of peptidases that presumably generate amino acids (Plamann et al., J. Bacteriol. 174:3311-3318 (1992)). A-factor is diffusible, and therefore does not require direct cell-to-cell contact for signal transmission. In contrast, C-Factor is normally found tightly associated with the cell surface of the signal producer, and transmission requires close contact between the signal producer and the recipient. Thus, C-signaling requires cellular motion and the close physical contact of the swarming cells in an aggregate. Both signal types provide the necessary format for the required message; A-factor to attract distant cells to a focus, and C-factor to maintain communication within the developing fruiting body. Each of the signals leads to a cascade of genetic switches that continues the cell differentiation process.
Many of the downstream regulatory and effecter genes have now been identified in M. xanthus using genetic and biochemical approaches, and it is the speed and efficiency with which bacteria allow analysis of the complex networks and metabolic pathways that provides a primary utility of the genome sequence.
The nucleic acid molecules and sequences disclosed herein represent a substantial portion of the M. xanthus genome. These molecules and sequences may be used to identify novel genes, for example genes involved in antibiotic production, and sequences in regulatory regions of the Myxococcus genes provided herein. The M. xanthus molecules and sequences also permit identification of genetic sequences from other organisms, including plants, mammals such as humans, bacteria, other filamentous fungi and non-filamentous fungi such as a yeast, e.g. by comparison of such sequences with M. xanthus sequences. The availability of a substantially complete set of genes or partial genes of the M. xanthus genome permits the definition of primers for fabricating representative nucleic acid molecules of the genome which can be used on microarrays to facilitate transcription profile studies. Such studies can help to identify regulatory networks and genes of interest in, for example, production of secondary metabolites, cell-to-cell signaling, cellular differentiation, and motility.
In addition, the M. xanthus genome fragments and sequences provided herein permit the fabrication of a wide variety of DNA constructs useful for imparting unique genetic properties into transgenic organisms. These and other advantages attendant with the various aspects of this invention will be apparent from the following description of the invention and its various embodiments.
The present invention contemplates and provides nucleic acid molecules comprising a substantial part of the genome of the bacterium Myxococcus xanthus. One aspect of the invention is a set of 1849 contig and singleton sequences comprising coding sequences, DNA replication elements, as well as promoters and other regulatory elements, such sequences being represented herein as SEQ ID NO: 1 through SEQ ID NO: 1849. Contigs in SEQ ID NO: 1 through SEQ ID NO: 1849 are recognized as those sequences whose designations begin with MYX10C. Singleton sequences are recognized as those having designations that begin with MYX10S. The present invention also encompasses complements of the nucleic acid sequences provided herein. Thus, a subset of the nucleic acid molecules of this invention comprises DNA protein encoding regions, replication elements, promoters and/or other regulatory elements of the M. xanthus genome as present in SEQ ID NO: 1 through SEQ ID NO 1849 or complements thereof.
Another aspect of this invention comprises a set of about 7842 genes or partial genes of the M. xanthus genome including genes represented by SEQ ID NO: 1850 through SEQ ID NO: 9691 and described in Table 1. As used herein, a substantially complete set of genes for an organism is referred to as a unigene set. Thus, as used herein reference is made to specific genes comprising the unigene set of M. xanthus as xe2x80x9cMYX12U_xxxxxe2x80x9d where MYX12U is an acronym for Myxococcus xanthus unigene and xxxx represents a number. Moreover, the term xe2x80x9cMYXUxe2x80x9d by itself is also used herein to mean any of the nucleic acid molecules comprising genes or partial genes of the unigene set for M. xanthus. More particularly the term xe2x80x9cMYXU of this inventionxe2x80x9d as used herein means a nucleic acid molecule representing a gene or partial gene of M. xanthus disclosed herein selected from the group consisting of SEQ ID NO: 1850 through SEQ ID NO: 9691. Preferred aspects of this invention contemplate MYXUs as identified by value of the gene prediction method, i.e., BLASTX or GeneMark. Certain preferred MYXUs have a BLASTX Bit Score of at least 100, more preferably 150. Other preferred MYXUs have a GeneMark Probability Score of at least 0.6, more preferably at least 0.75. Still other preferred MYXUs have a BLASTX Bit Score of at least 100 and a GeneMark Probability Score of 0.6; more preferably, respective scores of 150 and 0.75.
The present invention also contemplates and provides substantially purified nucleic acid molecules comprising the MYXUs and other nucleic acid molecules of this invention as well as molecules which are complementary to, and capable of specifically hybridizing to, an MYXU or its complement.
The present invention also contemplates and provides substantially purified nucleic acid molecules which are homologous to the nucleic acid molecules of this invention including, for example, those which are homologous to the MYXUs of this invention, e.g., a plurality of related sets of homologous nucleic acid molecules in other species which are homologous to the MYXUs.
The present invention also contemplates and provides substantially purified protein, or polypeptide fragments thereof, which are encoded by nucleic acid molecules of the present invention. Of particular interest is the group of 7134 Myxococcus proteins, peptides or fragments provided herein as SEQ ID NO: 9692 through SEQ ID NO:16825 and designated as MYX12_xxxx_prot, where xxxx is a number corresponding to the MYXU nucleic acid sequence which encodes the peptide.
The present invention also contemplates and provides constructs comprising DNA replication elements, promoters, regulatory elements and/or protein encoding regions that are useful in making transgenic cells or organisms. In particular this invention also provides transformed cells or organisms having a nucleic acid molecule which comprises: (a) a promoter region which functions in the cell to cause the production of an mRNA molecule, which is linked to (b) a structural nucleic acid molecule, which is linked to (c) a 3xe2x80x2 non-translated sequence that functions in the cell to cause termination of transcription, where components (a) and/or (b) are selected from M. xanthus nucleic acid sequences provided herein and more preferably where component (b) is selected from M. xanthus nucleic acid sequences which encode the peptide sequences, provided herein as SEQ ID NO:9692 through SEQ ID NO:16825.
Other aspects of this invention include oligonucleotides (and subsets thereof) for amplification or detection of the nucleic acid molecules of this invention. Such oligonucleotides may be used in analysis of Myxococcus gene expression patterns, either as probes or as elements on gene arrays, or to generate and isolate nucleic acid molecules representative of M. xanthus genes of this invention and homologs thereof in other myxobacteria species. Thus, the nucleic acids molecules of this invention including the oligonucleotides provided, represent a useful tool in genetic research not only for M. xanthus, but also for other bacterial species, particularly for other myxobacteria.
The present invention also contemplates and provides computer readable media having recorded thereon one or more of the nucleotide sequences provided by this invention and methods for using such media, e.g. in searching to identify genes associated with nucleic acid sequences.
The present invention also contemplates and provides collections of nucleic acid molecules, including oligonucleotides, representing the M. xanthus genome including collections on solid substrates, e.g. substrates having attached thereto in array form nucleic acid molecules or oligonucleotides representing genes of the M. xanthus genome. The invention also contemplates and provides methods of using such collections and arrays, e.g. in transcription profiling analysis. The present invention also contemplates and provides methods for using the nucleic acid molecules of this invention, e.g. for identifying genetic material and/or determining gene expression by hybridizing expressed and labeled nucleic acid molecules or fragments thereof to arrayed collections of the nucleic acid molecules of this invention.
The present invention also contemplates and provides oligonucleotides which are identical or complementary to a sequence of similar length in an MYXU. Such oligonucleotides are useful, for example, for hybridizing to and identifying nucleic acid molecules which are homologous and/or complementary to the MYXUs of the present invention.
Other aspects of this invention contemplate methods of using the MYXUs, e.g., for determining gene expression, for identifying mutations in a gene of interest and for constructing mutations in a gene of interest.